Mechanosensory signal transmission in the arms and the nerve ring, an interarm connective, of Octopus bimaculoides.

Octopuses coordinate their arms in a range of complex behaviors. In addition to brain-based sensorimotor integration and control, interarm coordination also occurs through a nerve ring at the arms' base. Here, we examine responses to mechanosensory stimulation of the arms by recording neural activity in the stimulated arm, the nerve ring, and other arms in a preparation of only the ring and arms. Arm axial nerve cords show graded responses to mechanosensory input and activity is transmitted proximally and distally in the arm. Mechanostimulation of one arm generates spiking in the nerve ring and in other arms. Activity in the nerve ring decreases with distance from the stimulated arm. Spontaneous activity with a range of spiking patterns occurs in the axial nerve cords and the nerve ring. These data show rich interarm signaling that supports arm control and coordination occurring outside of the brain.

Multiple nerve cords connect the arms of octopuses, providing alternative paths for inter-arm signaling.

Octopuses are remarkable in their ability to use many arms together during behavior (e.g., see Levy et al., 1 Mather,2 Byrne et al.,3 and Hanlon et al.4). Arm responses and multi-arm coordination can occur without engagement of major brain regions,5 which indicates the importance of local proprioceptive responses and peripheral connections. Here, we examine the intramuscular nerve cords (INCs),6,7,8,9 the key proprioceptive anatomy in the arms. INCs are understood to include proprioceptive neurons, multipolar neurons, and motoneurons (reviewed by Graziadei10) and are thought to contribute to structuring whole-arm movement.11 There are four INCs running the full length of each arm (e.g., see Guérin-Ganivet,6 Martoja and May,8 and Graziadei9); we focused on the pair closest to the suckers, called the oral INCs. In tracking the oral INCs, we found that they extend proximally and continue beyond the arm, through the arm's base. Each oral INC bypasses two adjacent arms and is continuous with the nearer oral INC of the third arm over. As a result, an arm connects through oral INC pathways to arms that are two arms away to the right and left of it. In addition to connecting distant arms, nerve fibers project from the central region of the INCs, suggesting function in local tissues. The other two INCs, paired aboral INCs, also extend proximally beyond the arm's base with trajectories suggestive of the oral INC pattern. These data identify previously unknown regions of the INCs that link distant arms, creating anatomical connections. They suggest potential INC proprioceptive function in extra-arm tissues and contribute to an understanding of embodied organization for octopus behavioral control.12,13,14,15.

Extraoral Taste Buds on the Paired Fins of Damselfishes.

Some fish species have taste buds on the surface of their bodies and fins, as well as in the oral cavity. The extraoral taste system of fish has traditionally been studied in species that inhabit environments and/or employ feeding strategies where vision is limited. Here we examined taste sensation in a new ecological context by investigating the paired fins of damselfish (Pomacentridae), a group of diurnal midwater fishes that inhabit the light-rich waters of coral reefs. Immunohistochemistry demonstrated the presence of taste buds on the paired fins of Chromis viridis, including on the distal tips of elongate leading-edge pelvic fin rays, where they are particularly densely packed, suggesting specialization for chemosensation. Similar anatomical results were also recorded from two other species, Pomacentrus amboinensis and Pomacentrus coelestis. We found that afferent pectoral fin nerves of C. viridis responded to a food-derived stimulus. By investigating the extraoral taste system in a new phylogenetic and ecological context, these results show that taste buds on fins are more widespread amongst fish than previously known and are present even in highly visual environments.

The Water to Land Transition Submerged: Multifunctional Design of Pectoral Fins for Use in Swimming and in Association with Underwater Substrate.

Fins of fishes provide many examples of structures that are beautifully designed to power and control movement in water; however, some species also use their fins for substrate-associated behaviors where interactions with solid surfaces are key. Here, we examine how the pectoral fins of ray-finned fish with these multifunctional behavioral demands, in water and on solid surfaces, are structured and function. We subdivide fins used in swimming and substrate contact into two general morphological categories, regionalized vs. generalized fins. Regionalized fins have ventral rays that are free from connecting membrane or in which that membrane is reduced. Dorsally they maintain a more typical membranous fin. While all pectoral fins vary somewhat in their morphology from leading to trailing edge, generalized fins do not have the substantial membrane loss between rays that is seen in regionalized fins and the distal edge anatomy changes gradually along its margin. We add a new case study in regionalized fins with the dwarf hawkfish (Cirrhitichthys falco). Hawkfishes are most often found perching and moving on structures in their environments. During perching, the free ventral rays are in contact with the substrate and splayed. We found that unlike other fish with regionalized pectoral fins, hawkfish maintain use of the dorsal membranous region of its pectoral fin for rhythmic swimming. We found that typically hawkfish bend their ventral free rays under, toward the medial hemitrichs or hold them straight during substrate-associated postures. This appears also to be the case for the ventral free rays of other species with regionalized fins. Generalized fin use for substrate contact was reviewed in round gobies (Neogobius melanostomus). In addition, although their lobe fins are not representative of ray-finned fish anatomy, we explored fin contact on submerged substrates in the Senegal bichir (Polypterus senegalus), which has a generalized distal fin (no free fin rays or distinct membrane regions). Both groups use their pectoral fins for swimming. During substrate-based postures, unlike hawkfish, their distal rays generally bend outward toward the lateral hemitrichs and a large swath of the fin membrane can contact the surface. The alternative demands on multifunctional fins suggest specialization of the mechanosensory system. We review mechanosensation related to fin movement and surface contact. These alternative regionalized and generalized strategies for serving aquatic and substrate-based functions underwater provide opportunities to further investigate specializations, including sensory structures and systems, that accompany the evolution of substrate-based behaviors in vertebrates.

Evolution of touch and proprioception of the limbs: Insights from fish and humans.

The function of the hands is inextricably linked to cutaneous mechanosensation, both in touch and in how hand movement and posture (proprioception) are controlled. The structure and behavior of hands and distal forelimbs of other vertebrates have been evolutionarily shaped by these mechanosensory functions. The distal forelimb of tetrapod vertebrates is homologous to the pectoral fin rays and membrane of fishes. Fish fins demonstrate similar mechanosensory abilities to hands and other distal tetrapod forelimbs in touch and proprioception. These results indicate that vertebrates were using the core mechanosensory inputs, such as fast adapting and slow adapting nerve responses, to inform fin and limb function and behavior before their diversification in fish and tetrapod lineages.

Morphological and physiological properties of Rohon-Beard neurons along the zebrafish spinal cord.

Primary mechanosensory neurons play an important role in converting mechanical forces into the sense of touch. In zebrafish, Rohon-Beard (RB) neurons serve this role at embryonic and larval stages of development. Here we examine the morphology and physiology of RBs in larval zebrafish to better understand how mechanosensory stimuli are represented along the spinal cord. We report that the morphology of RB neurons differs along the rostrocaudal body axis. Rostral RB neurons arborize in the skin near the cell body whereas caudal cells arborize at a distance posterior to their cell body. Using a novel electrophysiological approach, we also found longitudinal differences in the mechanosensitivity and physiological properties of RB neurons. Rostral RB neurons respond to mechanical stimulations close to the soma and produce up to three spikes with increasing stimulus intensity, whereas caudal cells respond at more distal locations and can produce four or more spikes when the intensity of the mechanical stimulus increases. The mechanosensory properties of RB neurons are consistent with those of rapidly adapting mechanoreceptors and can signal the onset, offset and intensity of mechanical stimulation. This is the first report of the intensity encoding properties of RB neurons, where an increase in spike number and a decrease in spike latency are observed with increasing stimulation intensity. This study reveals an unappreciated complexity of the larval zebrafish mechanosensory system and demonstrates how differences in the morphological and physiological properties of RBs related to their rostrocaudal location can influence the signals that enter the spinal cord.

Hindbrain and Spinal Cord Contributions to the Cutaneous Sensory Innervation of the Larval Zebrafish Pectoral Fin.

Vertebrate forelimbs contain arrays of sensory neuron fibers that transmit signals from the skin to the nervous system. We used the genetic toolkit and optical clarity of the larval zebrafish to conduct a live imaging study of the sensory neurons innervating the pectoral fin skin. Sensory neurons in both the hindbrain and the spinal cord innervate the fin, with most cells located in the hindbrain. The hindbrain somas are located in rhombomere seven/eight, laterally and dorsally displaced from the pectoral fin motor pool. The spinal cord somas are located in the most anterior part of the cord, aligned with myomere four. Single cell reconstructions were used to map afferent processes and compare the distributions of processes to soma locations. Reconstructions indicate that this sensory system breaks from the canonical somatotopic organization of sensory systems by lacking a clear organization with reference to fin region. Arborizations from a single cell branch widely over the skin, innervating the axial skin, lateral fin surface, and medial fin surface. The extensive branching over the fin and the surrounding axial surface suggests that these fin sensory neurons report on general conditions of the fin area rather than providing fine location specificity, as has been demonstrated in other vertebrate limbs. With neuron reconstructions that span the full primary afferent arborization from the soma to the peripheral cutaneous innervation, this neuroanatomical study describes a system of primary sensory neurons and lays the groundwork for future functional studies.

Sensing the structural characteristics of surfaces: texture encoding by a bottom-dwelling fish.

The texture of contacted surfaces influences our perception of the physical environment and modulates behavior. Texture perception and its neural encoding mechanisms have traditionally been studied in the primate hand, yet animals of all types live in richly textured environments and regularly interact with textured surfaces. Here we explore texture sensation in a different type of vertebrate limb by investigating touch and potential texture encoding mechanisms in the pectoral fins of fishes, the forelimb homologs. We investigated the pectoral fins of the round goby (Neogobius melanostomus), a bottom-dwelling species that lives on substrate types of varying roughness and whose fins frequently contact the bottom. Analysis shows that the receptive field sizes of fin ray afferents are small and afferents exhibit response properties to tactile motion that are consistent with those of primates and other animals studied previously. In response to a periodic stimulus (coarse gratings), afferents phase lock to the stimulus temporal frequency and thus can provide information about surface texture. These data demonstrate that fish can have the capability to sense the tactile features of their near range physical environment with fins.

DNA-based fluorescent probes of NOS2 activity in live brains.

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.

Pectoral fin kinematics and motor patterns are shaped by fin ray mechanosensation during steady swimming in Scarus quoyi.

For many fish species, rhythmic movement of the pectoral fins, or forelimbs, drives locomotion. In terrestrial vertebrates, normal limb-based rhythmic gaits require ongoing modulation with limb mechanosensors. Given the complexity of the fluid environment and dexterity of fish swimming through it, we hypothesize that mechanosensory modulation is also critical to normal fin-based swimming. Here, we examined the role of sensory feedback from the pectoral fin rays and membrane on the neuromuscular control and kinematics of pectoral fin-based locomotion. Pectoral fin kinematics and electromyograms of the six major fin muscles of the parrotfish, Scarus quoyi, a high-performance pectoral fin swimmer, were recorded during steady swimming before and after bilateral transection of the sensory nerves extending into the rays and surrounding membrane. Alternating activity of antagonistic muscles was observed and drove the fin in a figure-of-eight fin stroke trajectory before and after nerve transection. After bilateral transections, pectoral fin rhythmicity remained the same or increased. Differences in fin kinematics with the loss of sensory feedback also included fin kinematics with a significantly more inclined stroke plane angle, an increased angular velocity and fin beat frequency, and a transition to the body-caudal fin gait at lower speeds. After transection, muscles were active over a larger proportion of the fin stroke, with overlapping activation of antagonistic muscles rarely observed in the trials of intact fish. The increased overlap of antagonistic muscle activity might stiffen the fin system in order to enhance control and stability in the absence of sensory feedback from the fin rays. These results indicate that fin ray sensation is not necessary to generate the underlying rhythm of fin movement, but contributes to the specification of pectoral fin motor pattern and movement during rhythmic swimming.

Escape responses of fish: a review of the diversity in motor control, kinematics and behaviour.

The study of fish escape responses has provided important insights into the accelerative motions and fast response times of these animals. In addition, the accessibility of the underlying neural circuits has made the escape response a fundamental model in neurobiology. Fish escape responses were originally viewed as highly stereotypic all-or-none behaviours. However, research on a wide variety of species has shown considerable taxon-specific and context-dependent variability in the kinematics and neural control of escape. In addition, escape-like motions have been reported: these resemble escape responses kinematically, but occur in situations that do not involve a response to a threatening stimulus. This Review focuses on the diversity of escape responses in fish by discussing recent work on: (1) the types of escape responses as defined by kinematic analysis (these include C- and S-starts, and single- versus double-bend responses); (2) the diversity of neuromuscular control; (3) the variability of escape responses in terms of behaviour and kinematics within the context of predator-prey interactions; and (4) the main escape-like motions observed in various species. Here, we aim to integrate recent knowledge on escape responses and highlight rich areas for research. Rapidly developing approaches for studying the kinematics of swimming motion both in the lab and within the natural environment provide new avenues for research on these critical and common behaviours.

Comparative Principles for Next-Generation Neuroscience.

Neuroscience is enjoying a renaissance of discovery due in large part to the implementation of next-generation molecular technologies. The advent of genetically encoded tools has complemented existing methods and provided researchers the opportunity to examine the nervous system with unprecedented precision and to reveal facets of neural function at multiple scales. The weight of these discoveries, however, has been technique-driven from a small number of species amenable to the most advanced gene-editing technologies. To deepen interpretation and build on these breakthroughs, an understanding of nervous system evolution and diversity are critical. Evolutionary change integrates advantageous variants of features into lineages, but is also constrained by pre-existing organization and function. Ultimately, each species' neural architecture comprises both properties that are species-specific and those that are retained and shared. Understanding the evolutionary history of a nervous system provides interpretive power when examining relationships between brain structure and function. The exceptional diversity of nervous systems and their unique or unusual features can also be leveraged to advance research by providing opportunities to ask new questions and interpret findings that are not accessible in individual species. As new genetic and molecular technologies are added to the experimental toolkits utilized in diverse taxa, the field is at a key juncture to revisit the significance of evolutionary and comparative approaches for next-generation neuroscience as a foundational framework for understanding fundamental principles of neural function.

Adipose fin development and its relation to the evolutionary origins of median fins.

The dorsal, anal and caudal fins of vertebrates are proposed to have originated by the partitioning and transformation of the continuous median fin fold that is plesiomorphic to chordates. Evaluating this hypothesis has been challenging, because it is unclear how the median fin fold relates to the adult median fins of vertebrates. To understand how new median fins originate, here we study the development and diversity of adipose fins. Phylogenetic mapping shows that in all lineages except Characoidei (Characiformes) adipose fins develop from a domain of the larval median fin fold. To inform how the larva's median fin fold contributes to the adipose fin, we study Corydoras aeneus (Siluriformes). As the fin fold reduces around the prospective site of the adipose fin, a fin spine develops in the fold, growing both proximally and distally, and sensory innervation, which appears to originate from the recurrent ramus of the facial nerve and from dorsal rami of the spinal cord, develops in the adipose fin membrane. Collectively, these data show how a plesiomorphic median fin fold can serve as scaffolding for the evolution and development of novel, individuated median fins, consistent with the median fin fold hypothesis.

Fins as Mechanosensors for Movement and Touch-Related Behaviors.

Mechanosensation is a universal feature of animals that is essential for behavior, allowing detection of animals' own body movement and position as well as physical characteristics of the environment. The extraordinary morphological and behavioral diversity that exists across fish species provide rich opportunities for comparative mechanosensory studies in fins. The fins of fishes have been found to function as proprioceptors, by providing feedback on fin ray position and movement, and as tactile sensors, by encoding pressures applied to the fin surface. Across fish species, and among fins, the afferent response is remarkably consistent, suggesting that the ability of fin rays and membrane to sense deformation is a fundamental feature of fish fins. While fin mechanosensation has been known in select, often highly specialized, species for decades, only in the last decade have we explored mechanosensation in typical propulsive fins and considered its role in behavior, particularly locomotion. In this paper, we synthesize the current understanding of the anatomy and physiology of fin mechanosensation, looking toward key directions for research. We argue that a mechanosensory perspective informs studies of fin-based propulsion and other fin-driven behaviors and should be considered in the interpretation of fin morphology and behavior. In addition, we compare the mechanosensory system innervating the fins of fishes to the systems innervating the limbs of mammals and wings of insects in order to identify shared mechanosensory strategies and how different organisms have evolved to meet similar functional challenges. Finally, we discuss how understanding the biological organization and function of fin sensors can inform the design of control systems for engineered fins and fin-driven robotics.

A comparison of pectoral fin ray morphology and its impact on fin ray flexural stiffness in labriform swimmers.

The organization of tissues in appendages often affects their mechanical properties and function. In the fish family Labridae, swimming behavior is associated with pectoral fin flexural stiffness and morphology, where fins range on a continuum from stiff to relatively flexible fins. Across this diversity, pectoral fin flexural stiffness decreases exponentially along the length of any given fin ray, and ray stiffness decreases along the chord of the fin from the leading to trailing edge. In this study, we examine the morphological properties of fin rays, including the effective modulus in bending (E), second moment of area (I), segmentation, and branching patterns, and their impact on fin ray stiffness. We quantify intrinsic pectoral fin ray stiffness in similarly sized fins of two closely related species that employ fins of divergent mechanics, the flapping Gomphosus varius and the rowing Halichoeres bivittatus. While segmentation patterns and E were similar between species, measurements of I and the number of fin ray branch nodes were greater in G. varius than in H. bivittatus. A multiple regression model found that of these variables, I was always significantly correlated with fin ray flexural stiffness and that variation in I always explained the majority of the variation in flexural stiffness. Thus, while most of the morphological variables quantified in this study correlate with fin ray flexural stiffness, second moment of area is the greatest factor contributing to variation in flexural stiffness. Further, interspecific variation in fin ray branching pattern could be used as a means of tuning the effective stiffness of the fin webbing to differences in swimming behavior and hydrodynamics. The comparison of these results to other systems begins to unveil fundamental morphological features of biological beams and yields insight into the role of mechanical properties in fin deformation for aquatic locomotion.

The relationship between pectoral fin ray stiffness and swimming behavior in Labridae: insights into design, performance and ecology.

The functional capabilities of flexible, propulsive appendages are directly influenced by their mechanical properties. The fins of fishes have undergone extraordinary evolutionary diversification in structure and function, which raises questions of how fin mechanics relate to swimming behavior. In the fish family Labridae, pectoral fin swimming behavior ranges from rowing to flapping. Rowers are more maneuverable than flappers, but flappers generate greater thrust at high speeds and achieve greater mechanical efficiency at all speeds. Interspecific differences in hydrodynamic capability are largely dependent on fin kinematics and deformation, and are expected to correlate with fin stiffness. Here we examine fin ray stiffness in two closely related species that employ divergent swimming behaviors, the flapping Gomphosus varius and the rowing Halichoeres bivittatus To determine the spatial distribution of flexural stiffness across the fin, we performed three-point bending tests at the center of the proximal, middle and distal regions of four equally spaced fin rays. Pectoral fin ray flexural stiffness ranged from 0.0001 to 1.5109 µN m2, and the proximal regions of G. varius fin rays were nearly an order of magnitude stiffer than those of H. bivittatus In both species, fin ray flexural stiffness decreased exponentially along the proximodistal span of fin rays, and flexural stiffness decreased along the fin chord from the leading to the trailing edge. Furthermore, the proportion of fin area occupied by fin rays was significantly greater in G. varius than in H. bivittatus, suggesting that the proportion of fin ray to fin area contributes to differences in fin mechanics.

Mechanosensation is evolutionarily tuned to locomotor mechanics.

The biomechanics of animal limbs has evolved to meet the functional demands for movement associated with different behaviors and environments. Effective movement relies not only on limb mechanics but also on appropriate mechanosensory feedback. By comparing sensory ability and mechanics within a phylogenetic framework, we show that peripheral mechanosensation has evolved with limb biomechanics, evolutionarily tuning the neuromechanical system to its functional demands. We examined sensory physiology and mechanics of the pectoral fins, forelimb homologs, in the fish family Labridae. Labrid fishes exhibit extraordinary morphological and behavioral diversity and use pectoral fin-based propulsion with fins ranging in shape from high aspect ratio (AR) wing-like fins to low AR paddle-like fins. Phylogenetic character analysis demonstrates that high AR fins evolved independently multiple times in this group. Four pairs of species were examined; each included a plesiomorphic low AR and a high AR species. Within each species pair, the high AR species demonstrated significantly stiffer fin rays in comparison with the low AR species. Afferent sensory nerve activity was recorded during fin ray bending. In all cases, afferents of stiffer fins were more sensitive at lower displacement amplitudes, demonstrating mechanosensory tuning to fin mechanics and a consistent pattern of correlated evolution. We suggest that these data provide a clear example of parallel evolution in a complex neuromechanical system, with a strong link between multiple phenotypic characters: pectoral fin shape, swimming behavior, fin ray stiffness, and mechanosensory sensitivity.

Local Spinal Cord Circuits and Bilateral Mauthner Cell Activity Function Together to Drive Alternative Startle Behaviors.

The reticulospinal Mauthner cells (M-cells) of the startle circuit have been considered to be dedicated to one basic motor output and the C-type startle response in fish. The neural circuit underlying the C-start, a startle behavior in which the fish forms a "C"-shaped body bend has been described in depth in goldfish and zebrafish [1, 2] and is thought to occur in other species [3, 4]. However, previous research has shown that some species can perform a second type of startle called the S-start [5-7]. This startle response, in which the first movement creates an "S"-shaped body bend achieved with regional muscle activity on left and right sides, cannot be explained by M-cell circuit models. Here we use larval zebrafish to examine the S-start circuit. Since S-starts are elicited through tail stimulation [5-7] and ablating M-cells abolishes short-latency tail-elicited startles [8, 9], we hypothesized that M-cell activity was necessary for S-start generation. Our findings show that the M-cells fire simultaneously to generate the S-start. However, simultaneous M-cell spikes generated through direct current injection were not sufficient to generate S-starts. Through recordings of motoneurons, inhibitory interneurons, and sensory neurons, we uncover a mechanism for generating alternative startle behaviors; local sensory inputs drive inhibitory interneuron activity, which inhibits caudal motoneurons and pre-conditions their excitability prior to the arrival of M-cell spikes in the tail. We suggest that this motoneuron hyperpolarization can bias motor output to left or right sides, determining whether the fish performs a C-start or an S-start behavior.